Diaminopimelete epimerase catalyzes the interconversion of L,L-and D,L-meso-diaminopimelate, via Calpha proton abstraction from one isomer and protonation at the opposite face of the carbanionic intermediate or transition state. While no structures of other members of the PLP-independent amino acid racemase family have yet been determined, the dapF encoded enzyme from Haemophilus influenzae has been cloned, expressed, purified and crystallized, and the three- dimensional structure was solved to 2.7 A. This 274 amino acid enzyme exhibits a novel fold, and the two catalytic cysteine residues, Cys73 and Cys217, are located in a cleft separating the two structurally homologous domains. The proposed kinetic and isotopic studies of the single mutants, C73S and C217S, will further characterize the unusual role of the cysteine as a general acid and base (versus its more typical role as a nucleophile in other enzyme catalyzed reactions). These experiments will also assign which cysteine residue is responsible for proton abstraction in the L, L- >D,L and D,L->L,L directions, respectively. In addition, the measurement of the microscopic pK values of the two cysteine residues, Cys73 and Cys217, using specific isotopic labeling and 13C nuclear magnetic resonance, will reveal details of the active site environment and will clarify the recent results from the steady-stage pH studies. Further, while no enzyme-substrate or enzyme-inhibitor structure is available, the substrate-binding site will also be analyzed using hydrogen/deuterium amide exchange and mass spectrometry. These proposed experiments will further understanding of the mechanism of diaminopimelate epimerase which is unusual among the cofactor independent racemases with unique properties including an equilibrium constant equal to 2.